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The state of an electrode surface can have dramatic effects on the oxidation-reduction activity of solution species. Selective modification of the surface enables control of these effects. One common method of electrode modification is covalent attachment of selected functional groups via phenyl diazonium ion reduction. The result is a near monomolecular layer of specific molecules at the electrode surface. The chemical functionality of the diazonium ion is the selectivity component of the electrode. Much electrode modification research has used para-substitution of the phenyl ring. An intriguing question is whether asymmetric polar molecules (ortho- or meta- substitution of the phenyl ring) produces unique electrochemical results due to steric hindrances and/or molecular alignment of the molecules on the electrode surface as a result of polarity differences. The synthesis, isolation, and characterization of five diazonium ions; one nonpolar [benzene diazonium], one polar symmetric [4-nitrobezene diazonium], and three polar asymmetric [2-nitrobenzene diazonium, 3-(trifluoromethyl) benzene diazonium, and 2-(trifluoromethoxy) benzene diazonium]; isolated as tetrafluoroborate salts from their amine precursors is presented. Electrochemical properties of the synthesized diazonium ions towards surface modification of glassy carbon electrodes are compared based on functionality. Further, electrochemical activity model systems on the modified electrodes are compared.
In the halophilic archaeon Haloferax volcanii, tRNA intron endonuclease is known to cleave precursor tRNAs at specific bulge-helix-bulge (BHB) motifs. In this report, intron endonuclease has been over expressed in Haloferax volcanii (Hvo.) using a novel inducible expression system controlled by the histidine utilization (Hut) operon. Protein expression is induced by addition of urocanate, a metabolite of histidine. The native intron endonuclease catalyzes intron cleavage of pre-tRNA-Trp and pre-tRNA-Gln(CCA) more efficiently than the enzyme expressed in E. coli. This is not surprising since the E. coli expressed enzyme requires refolding with urea and the Hvo. expressed enzyme is soluble. Optimal cleavage conditions are the same for the enzyme from both sources: 40 mM Tris-HCl pH 7.4, 2% glycerol, 20 mM MgCl2 and 2 M KCl. Intron endonuclease isolated from Hvo. will improve understanding of tRNA processing by this enzyme. The Hut expression system is a new mechanism to produce halophilic proteins.
Riboflavin is a micronutrient required for maintaining health. Riboflavin, also known as vitamin B2 or additive E101, is important in energy metabolism and the metabolism of fats, ketone bodies, carbohydrates, and proteins. Riboflavin binding protein (RBP) is a critical protein found in the yolks and whites of bird eggs that transports riboflavin from the mother bird to the developing embryo. Riboflavin is yellow and it is the large concentrations of this vitamin that make egg yolks and whites yellow. Previous student research projects have developed a purification protocol for RBP and an unfolding assay using fluorescence. There were two goals for this project. The first was to develop a protocol that can be used at Capital to determine the stoichiometric ratio of riboflavin to RBP. The second goal was to develop a protocol that will determine the unfolding characteristics of RBP using UV-Vis spectrophotometry.
Chromate ion (CrO42-) is used extensively as a corrosion inhibitor for many different applications. The mechanism of inhibition involves reduction to chromium [III] ion and strong attachment to the surface of the substrate. The layers formed are exceedingly thin, less than 10 nanometers, but the immobilized chromium [III] ion strongly resists electron transfer thereby inhibiting further electrochemical reactions. Using this property, a controlled deposition of chromate has been achieved on glassy carbon electrode material that partially inhibits the electrode surface by forming an incomplete barrier. As the surface coverage of chromium [III] increases, the remaining active areas of the electrode surface become smaller and more isolated from one another; beginning to behave as microelectrodes. These active areas ultimately form a random microelectrode array on the electrode surface and show altered voltammetric response towards model electrochemical systems as compared to a macroelectrode. Depending on experimental time scale, the array can exhibit semi-infinite linear or radial diffusion or a hybrid of the two. Conditions for optimal microelectrode array formation are presented including variations with chromate concentration, exposure time, and reducing potential. An assessment of electrode coverage and observations of microelectrode array behavior is shown.
Huntington’s Disease (HD) is a progressive neurological disorder that causes uncontrolled movements, depression, and a loss of cognition. HD is genetically inherited in an autosomal dominant pattern and is caused by a mutation of a CAG trinucleotide repeat in the HTT gene. In a normal HTT gene, the CAG segment is repeated 10-35 times and HTT successfully codes for huntingtin protein, however, if HD is present the CAG segment is repeated 36-120 times and an abnormally long huntingtin protein is produced. The elongated huntingtin protein is cut into smaller fragments that accumulate in neurons disrupting cell function; this has a particularly dramatic effect on the basal ganglia region of the brain. Digital media, painting, and pastels were used to portray the drastic physical and emotional impact caused by HD. By composing a unified visual presentation, a bridge between art and biochemical processes was constructed.
Flavodoxins are small bacterial flavoproteins that contain a noncovalently bound flavin mononucleotide (FMN). Flavodoxins catalyze reactions that are vital for metabolic pathways such as photosynthesis and the oxidation of pyruvate, and serve as useful model systems for studying more complex flavoproteins. In order to study the binding of FMN to flavodoxin, an attempt was made to make the S58C mutant. To make this mutation, site-directed mega-primer polymerase chain reaction (PCR) was used. The gene fragment was isolated using electrophoresis and gel extraction. The correct length of DNA, however, was not obtained. After repeating the experiment and altering different variables it is still uncertain why the mutant gene fragment was not made.
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